Significant advances in our understanding of renal transport physiology have traditionally depended on the development of novel experimental methods. Ussing and Leaf's pioneering use of planar anuran epithelial membranes, Burg's development of tubular microperfusion technology and Morrel's innovative micro-analytic methods profoundly expanded our understanding of the nephron. Increasingly, attention is being given to the application of cell culture technology to renal transport and endocrinology. Cell culture not only offers a new technical approach to long-standing problems but, more importantly, provides a tool for expanding renal physiology beyond traditional conceptional boundries. Renal physiology, placed in the theoretical and technical framework of cell and molecular biology acquires a plethora of novel directions and insights as exemplified by the transformation of classical neurophysiology into contemporary molecular neurobiology. For renal cell culture to have this impact, long-term functional cells lines derived from identified nephron segments must be available. Previous work has not succeeded in defining culture systems capable of sustaining extended growth and function in vitro because: 1. cultured cells were treated as independent microorganisms instead of units of interacting cells and 2. cells were forced to adapt to existing culture methods instead of designing novel methodology to support the needs of specialized epithelial cells. The principal goal of this project is to develop a basic understanding of the cultured renal epithelial cell. This will allow the non-empirical definition of a milieu capable of supporting functional epithelium in long-term culture. This project centers on the thick ascending limb, a nephron segment crucial to the control of extracellular fluid volume and a target of diuretics used in controlling essential hypertension. Understanding the molecular details of thick limb function provides a basis not only for developing more effective therapeutic modalities but may also help define the primary cellular defect in essential hypertension.